MTA2 Antibody

What is MTA2 protein and why is it a significant research target?

MTA2 (Metastasis-associated protein 2), also known as MTA1L1 or PID, is a 668 amino acid protein with a molecular weight of approximately 75 kDa. It plays a crucial role in gene expression regulation as a component of the nucleosome remodeling and histone deacetylation (NURD/NuRD) complex . The significance of MTA2 as a research target stems from its involvement in ATP-dependent chromatin remodeling and transcriptional repression, influencing cellular processes including differentiation and proliferation . MTA2's ability to modulate chromatin structure and gene expression makes it important in both normal cellular function and disease states, particularly in cancer progression .

What are the optimal antibody types for different MTA2 detection methods?

For optimal results, consider the following methodological guidelines:

• For WB applications requiring high sensitivity, mouse monoclonal antibodies (66195-1-Ig) provide superior results with less background at higher dilutions

• For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may be used as an alternative

• For multiplex applications, select antibodies from different host species to prevent cross-reactivity

How should I optimize MTA2 antibody concentration for Western blotting?

Optimizing antibody concentration for Western blotting requires a systematic approach:

• Start with a medium dilution range (e.g., 1:5000) from the manufacturer's recommendation

• Perform a dilution series experiment: For polyclonal antibodies (17554-1-AP), test dilutions between 1:2000-1:10000; for monoclonal antibodies (66195-1-Ig), test between 1:5000-1:50000

• Evaluate signal-to-noise ratio at each concentration

• Consider the cell/tissue type being analyzed - different samples may require different optimal concentrations

• For weak signals, particularly with endogenous MTA2, lower dilutions (higher antibody concentration) may be necessary, but monitor background

• Implement proper blocking (5% non-fat milk in TBS is effective)

• Secondary antibody optimization is also crucial - for rabbit primaries, goat anti-rabbit IgG-HRP at 1:5000 provides good results

Methodological observation: Mouse monoclonal antibodies typically allow for higher dilutions while maintaining specificity, which can be cost-effective for long-term research projects .

What positive controls are recommended for validating MTA2 antibody specificity?

Based on validated research data, the following positive controls are recommended for MTA2 antibody validation:

For proper validation methodology:

• Include at least one well-characterized positive control in each experiment

• Load approximately 50 μg of protein lysate per lane for optimal detection

• Compare observed molecular weight (70-75 kDa) with predicted weight (75 kDa)

• For definitive validation, include MTA2 knockdown/knockout samples as negative controls

• For tissue samples, confirm appropriate antigen retrieval method before concluding negative results

How can MTA2 antibodies be employed to study its interaction with the NURD/NuRD complex?

MTA2 functions as a key component of the nucleosome remodeling and histone deacetylation (NURD/NuRD) complex. To study these interactions:

• Co-Immunoprecipitation (Co-IP):

  • Use MTA2 antibodies (such as 17554-1-AP) for IP at 0.5-4.0 μg per 1-3 mg of protein lysate

  • Probe for other NURD/NuRD components (HDAC1/2, Mi-2, RbAp46/48)

  • Cross-validate by performing reverse Co-IP with antibodies against other complex members

• Chromatin Immunoprecipitation (ChIP):

  • MTA2 antibodies have been validated for ChIP applications

  • Follow with qPCR or sequencing to identify genomic binding regions

  • Compare MTA2 binding profiles with other NURD/NuRD components to identify co-occupancy

• Proximity Ligation Assay (PLA):

  • Combine MTA2 antibody with antibodies against other complex components

  • This technique visualizes protein-protein interactions in situ with high specificity

• Sequential ChIP (Re-ChIP):

  • First ChIP with MTA2 antibody followed by second ChIP with antibodies against other NURD/NuRD components

  • Identifies genomic regions where multiple complex members co-localize

Methodological consideration: When studying protein-protein interactions, antibody orientation is crucial - the epitope recognized by the antibody should not interfere with the interaction interface of interest .

What methodologies are recommended for investigating MTA2's role in p53 deacetylation and cancer progression?

MTA2 has been shown to deacetylate p53, affecting p53-dependent transcriptional activation and modulating cell growth arrest and apoptosis. Here are methodological approaches to investigate this relationship:

• Acetylation Status Analysis:

  • Immunoprecipitate p53 using specific antibodies

  • Probe with acetylation-specific antibodies (e.g., anti-acetyl-p53)

  • Compare acetylation levels in cells with normal vs. altered MTA2 expression

• MTA2 Modulation:

  • Use siRNA/shRNA for MTA2 knockdown (validated in multiple publications)

  • Alternatively, employ CRISPR-Cas9 for MTA2 knockout

  • Measure changes in p53 acetylation, activity, and downstream targets

• Functional Assays:

  • Cell cycle analysis: Flow cytometry to determine if MTA2 manipulation alters p53-dependent cell cycle arrest

  • Apoptosis assays: Annexin V staining, TUNEL assay to measure p53-dependent apoptosis

  • Reporter assays: Use p53-responsive luciferase reporters to measure transcriptional activity

• In vivo Models:

  • Immunohistochemistry to correlate MTA2 and p53 expression/activity in tumor samples

  • Use MTA2 antibodies (17554-1-AP or 66195-1-Ig) at 1:500-1:2000 dilutions

  • Compare with markers of tumor progression and metastasis

• Clinical Correlation:

  • Analyze MTA2 expression in patient samples using validated antibodies

  • Correlate with p53 mutation status, acetylation levels, and clinical outcomes

Technical note: When performing IHC on clinical samples, antigen retrieval with TE buffer (pH 9.0) provides optimal results, though citrate buffer (pH 6.0) may be used as an alternative .

How can MTA2 antibodies be utilized in multiplexed imaging systems for studying spatial organization in tissues?

Advanced multiplexed imaging techniques allow for simultaneous visualization of multiple proteins in their spatial context. For MTA2 studies:

• Multiplex Immunofluorescence:

  • Use mouse monoclonal MTA2 antibody (66195-1-Ig) at 1:1000-1:2000 dilution

  • Combine with antibodies against other proteins of interest from different host species

  • Select fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • For nuclear proteins like MTA2, confocal microscopy provides superior resolution

• Cyclic Immunofluorescence (CycIF):

  • Apply MTA2 antibody in one cycle, image, then strip/quench fluorescence

  • Repeat with different antibodies in subsequent cycles

  • Enables visualization of >30 proteins on the same sample

  • Critical for studying MTA2 in relation to multiple signaling pathways

• Mass Cytometry Imaging (IMC):

  • Label MTA2 antibodies with rare earth metal isotopes

  • Allows for simultaneous detection of >40 proteins with minimal signal overlap

  • Particularly useful for tumor microenvironment studies

• Proximity Ligation Assay (PLA) in Tissue Sections:

  • Combine MTA2 antibody with antibodies against potential interacting partners

  • Provides spatial information about protein-protein interactions in situ

Methodological considerations:

• For multiplexed approaches, validate antibodies individually before combining

• Include appropriate controls for each antibody in the panel

• MTA2 antibodies from mouse hosts (66195-1-Ig) work well in multiplexed systems with rabbit antibodies against other targets

• Counterstain nuclei with DAPI to facilitate identification of MTA2-positive nuclei

How can I resolve inconsistent MTA2 antibody staining patterns in immunohistochemistry?

Inconsistent staining patterns in IHC can arise from multiple factors. Here's a systematic troubleshooting approach:

• Antigen Retrieval Optimization:

  • MTA2 antibodies show optimal performance with TE buffer pH 9.0

  • If results are inconsistent, test alternative methods:

    • Citrate buffer pH 6.0 as an alternative

    • Experiment with different retrieval times (15-30 minutes)

    • Test both pressure cooker and water bath methods

• Fixation Variables:

  • Overfixation can mask epitopes - standardize fixation times

  • For FFPE tissues, section thickness should be consistent (4-5 μm)

  • Consider testing different fixatives if possible

• Antibody Dilution Series:

  • Test a wider range of dilutions than recommended:

    • For 17554-1-AP: 1:200, 1:500, 1:1000, 1:2000, 1:5000

    • For 66195-1-Ig: 1:250, 1:500, 1:1000, 1:2000, 1:4000

  • Different tissue types may require different optimal dilutions

• Detection System Evaluation:

  • Compare different visualization methods:

    • DAB-based detection systems (brown signal)

    • AP-based systems (red signal) may give cleaner results in some tissues

  • For fluorescent detection, test different fluorophores and mounting media

• Background Reduction:

  • Increase blocking time (60 minutes with 10% goat serum)

  • Add protein blockers (0.1% BSA) to antibody diluent

  • Include 0.1-0.3% Triton X-100 for improved penetration

Methodological note: When comparing staining patterns between different antibodies, process slides in parallel under identical conditions to minimize technical variability .

What strategies can address weak or absent MTA2 signal in Western blotting?

When encountering weak or absent MTA2 signals in Western blotting, consider these methodological interventions:

• Sample Preparation Optimization:

  • Use RIPA buffer with protease inhibitors for efficient extraction

  • For nuclear proteins like MTA2, ensure proper nuclear lysis

  • Avoid repeated freeze-thaw cycles of samples

  • Load adequate protein (50 μg recommended)

• Transfer Efficiency Improvement:

  • For 75 kDa proteins like MTA2, optimize transfer conditions:

    • Use wet transfer systems for higher efficiency

    • Transfer at 150mA for 50-90 minutes

    • Consider adding SDS (0.1%) to transfer buffer for better transfer of larger proteins

• Antibody Selection and Handling:

  • Try alternative antibodies if available

  • For polyclonal antibodies (17554-1-AP), use lower dilutions (1:2000)

  • For monoclonal antibodies (66195-1-Ig), test 1:5000 dilution

  • Ensure antibodies haven't degraded (store at -20°C, avoid repeated freeze-thaw)

• Signal Enhancement Techniques:

  • Use high-sensitivity ECL substrates

  • Increase exposure time incrementally

  • For weak signals, consider HRP-conjugated secondary antibodies with higher activity

• Membrane Handling:

  • Nitrocellulose membranes provide good results for MTA2 detection

  • Block thoroughly (5% non-fat milk in TBS for 1.5 hours)

  • Optimize incubation time (overnight at 4°C for primary antibody)

• Positive Control Inclusion:

  • Always include known positive controls (HeLa, HEK-293 cells)

  • If signal persists in controls but not in samples, expression level may be biological

Experimental observation: MTA2 signal can sometimes appear as a doublet around 70-75 kDa, representing different post-translational modification states or isoforms .

How can I differentiate between MTA2 and other MTA family members in my experiments?

The MTA family includes MTA1, MTA2, and MTA3, which share significant sequence homology. To ensure specificity for MTA2:

• Antibody Selection Criteria:

  • Choose antibodies specifically validated for MTA2 specificity

  • Some antibodies have been explicitly tested for lack of cross-reactivity with MTA1 (e.g., QED Bioscience 56227)

  • Examine the immunogen sequence - antibodies raised against unique regions have higher specificity

• Western Blot Differentiation:

  • MTA family members have different molecular weights:

    • MTA1: ~80 kDa

    • MTA2: 70-75 kDa

    • MTA3: ~65 kDa

  • Run controls expressing different MTA proteins to confirm specificity

  • Consider using gradient gels (5-20%) for better separation

• qPCR Validation:

  • Complement protein-level data with mRNA analysis

  • Design primers specific to unique regions of MTA2

  • Use this as orthogonal validation of antibody specificity

• Knockout/Knockdown Controls:

  • Include MTA2 knockdown/knockout samples

  • If the signal persists in knockout samples, cross-reactivity may be occurring

• Epitope Mapping:

  • Review the immunogen information:

    • C-terminal epitopes (aa 652-668) tend to be more specific to MTA2

    • Middle region antibodies may have higher cross-reactivity

• Immunoprecipitation-Mass Spectrometry:

  • For definitive identification, perform IP with MTA2 antibody

  • Analyze immunoprecipitated proteins by mass spectrometry

  • This can confirm the presence of MTA2 and detect potential cross-reactivity

Technical consideration: For applications requiring absolute specificity, monoclonal antibodies generally offer better discrimination between MTA family members than polyclonal antibodies .

How should researchers interpret variable MTA2 expression levels across different tissue types?

When analyzing MTA2 expression patterns across tissues, consider these methodological approaches and interpretive frameworks:

• Baseline Expression Profile:

  • MTA2 is widely expressed across tissues but with variable levels

  • Validated positive tissues include:

    • Mouse thymus

    • Human heart

    • Rat lung

    • Mouse brain

  • Establish reference expression levels in these tissues before comparing experimental samples

• Normalization Strategies:

  • For Western blot analysis:

    • Normalize to total protein (Ponceau S or REVERT staining)

    • Use multiple housekeeping proteins as loading controls

    • Consider nuclear-specific loading controls for nuclear proteins like MTA2

  • For IHC/IF quantification:

    • Express as percentage of positive cells

    • Use H-score or Allred scoring systems for semi-quantitative assessment

    • Employ digital image analysis for objective quantification

• Biological Interpretation Framework:

  • Higher expression in specific tissues may indicate:

    • Tissue-specific regulatory functions

    • Association with proliferative capacity of the tissue

    • Involvement in tissue-specific developmental programs

  • Changes during disease states may reflect:

    • Altered chromatin regulation

    • Modified transcriptional programs

    • Response to cellular stress

• Technical Variables to Consider:

  • Different fixation methods across tissue types can affect epitope availability

  • Tissue-specific protein extraction efficiency may vary

  • Endogenous peroxidase activity varies by tissue (relevant for IHC)

• Validation Through Orthogonal Methods:

  • Confirm protein-level observations with mRNA analysis

  • Compare antibody-based detection with reporter systems

  • Cross-validate with public databases (Human Protein Atlas, TCGA)

Methodological note: For accurate comparison across tissues, process all samples simultaneously using identical protocols, and include internal reference tissues in each experiment .

What are the key considerations when analyzing MTA2 localization in subcellular compartments?

MTA2 functions primarily in the nucleus as part of chromatin-modifying complexes, but proper analysis of its subcellular localization requires careful consideration:

• Fixation and Permeabilization Optimization:

  • For nuclear proteins like MTA2:

    • Paraformaldehyde (4%) fixation preserves nuclear architecture

    • Methanol fixation can improve nuclear antigen accessibility

    • Permeabilization is crucial - test Triton X-100 (0.1-0.5%) or saponin (0.1%)

• Counterstaining Strategy:

  • Always include nuclear counterstain (DAPI) for reference

  • Consider additional markers:

    • Nuclear membrane (Lamin B)

    • Nucleoli (Fibrillarin)

    • Heterochromatin (H3K9me3)

    • Euchromatin (H3K4me3)

• Quantitative Analysis Approaches:

  • Measure nuclear:cytoplasmic ratio of MTA2 signal

  • Assess co-localization with other nuclear proteins (Pearson's correlation)

  • Evaluate subnuclear distribution patterns (diffuse vs. punctate)

  • Consider 3D reconstruction from confocal z-stacks for complete analysis

• Dynamic Localization Considerations:

  • MTA2 localization may change with:

    • Cell cycle stage

    • Differentiation status

    • Stress conditions

    • Treatment with chromatin-modifying agents

  • Time-course experiments may reveal regulatory mechanisms

• Technical Controls for Localization Studies:

  • Include proteins with known localization patterns

  • Test multiple antibodies targeting different epitopes

  • Verify with fluorescent protein-tagged MTA2 constructs

  • Use fractionation followed by Western blot as orthogonal validation

Methodological insight: For immunofluorescence applications, confocal microscopy with deconvolution provides superior resolution of nuclear proteins like MTA2, particularly when examining co-localization with other nuclear factors .

How can researchers distinguish between technical artifacts and true biological variability in MTA2 immunostaining?

Discriminating between technical artifacts and genuine biological variability requires systematic controls and analysis:

• Comprehensive Control System:

  • Positive technical controls:

    • Known positive cell lines (HeLa, HEK-293)

    • Known positive tissues (thymus, heart)

  • Negative technical controls:

    • Primary antibody omission

    • Isotype control antibodies

    • Blocking peptide competition

  • Biological validation controls:

    • MTA2 knockdown/knockout samples

    • Tissues with known differential expression

• Batch Effect Minimization:

  • Process all comparative samples in parallel

  • Maintain identical conditions for all technical steps

  • Include standard reference samples across batches

  • Consider multiplexed approaches to reduce inter-slide variability

• Pattern Recognition Framework:

  • Technical artifacts often present as:

    • Edge effects or drying artifacts

    • Uniform background across all cell types

    • Signal in unexpected subcellular locations

  • Biological patterns typically show:

    • Cell type-specific expression

    • Correlation with differentiation/proliferation status

    • Appropriate subcellular localization (nuclear for MTA2)

    • Correlation with functionally related proteins

• Orthogonal Validation Strategies:

  • Confirm key findings with alternative antibodies

  • Validate with non-antibody methods (RNA-seq, reporter assays)

  • Compare with public database information

  • Use multiple detection methods (IF vs. IHC vs. WB)

• Quantitative Analysis Approaches:

  • Establish objective scoring criteria before analysis

  • Use digital image analysis when possible

  • Apply statistical methods appropriate for the data distribution

  • Consider blinded assessment by multiple observers

Technical insight: When troubleshooting inconsistent results, systematically vary one parameter at a time (fixation, antigen retrieval, antibody concentration) while keeping others constant to identify the source of variability .

How can MTA2 antibodies be employed in single-cell protein analysis technologies?

As single-cell technologies revolutionize biological research, MTA2 antibodies can be integrated into these emerging platforms:

• Single-Cell Western Blotting:

  • Adapting traditional Western blot to single-cell resolution

  • Requires highly specific antibodies with minimal background

  • MTA2 monoclonal antibodies (66195-1-Ig) are ideal candidates due to high specificity

  • Enables correlation of MTA2 expression with cellular heterogeneity

• Mass Cytometry (CyTOF):

  • MTA2 antibodies can be conjugated to rare earth metals

  • Allows simultaneous detection of MTA2 with >40 other proteins

  • Critical for understanding MTA2 in complex cellular hierarchies

  • No fluorescence overlap issues compared to flow cytometry

• Single-Cell Proteomics with Antibody-Based Capture:

  • MTA2 antibodies immobilized on microwell arrays

  • Capture proteins from lysed single cells

  • Detection via complementary antibodies or mass spectrometry

  • Reveals MTA2 protein interaction networks at single-cell resolution

• In situ Protein Analysis:

  • Techniques like CODEX use DNA-barcoded antibodies for spatial proteomics

  • MTA2 antibodies can be incorporated into multiplexed panels

  • Preserves tissue architecture while achieving single-cell resolution

  • Particularly valuable for understanding MTA2 in tumor microenvironments

• Proximity Ligation Assay at Single-Cell Level:

  • Combine MTA2 antibodies with antibodies against interaction partners

  • Detect specific protein-protein interactions in individual cells

  • Quantify interaction frequencies across heterogeneous populations

Methodological consideration: For single-cell applications, antibody validation is even more critical - perform extensive specificity testing, including isotype controls and blocking peptide competition assays before application in these sensitive techniques .

What are the methodological considerations for studying post-translational modifications of MTA2?

MTA2 function is regulated by various post-translational modifications (PTMs). Studying these modifications requires specialized approaches:

• Modification-Specific Antibody Selection:

  • For known MTA2 PTMs (phosphorylation, acetylation, SUMOylation)

  • Requires validation of modification-specific antibodies

  • Consider generating custom antibodies if commercial options unavailable

• Enrichment Strategies:

  • Immunoprecipitate total MTA2 using validated antibodies (17554-1-AP at 0.5-4.0 μg)

  • Follow with Western blotting using modification-specific antibodies

  • Alternative approach: enrich for specific modifications first, then probe for MTA2

• Mass Spectrometry Workflow:

  • Immunoprecipitate MTA2 with validated antibodies

  • Perform tryptic digestion

  • Analyze by LC-MS/MS with PTM-specific detection methods

  • Consider enrichment steps for low-abundance modifications

• Functional Correlation Analysis:

  • Compare PTM status across different:

    • Cell types

    • Cellular conditions (stress, differentiation)

    • Disease states

  • Correlate with MTA2 activity measurements

  • Test effects of PTM-mimicking or PTM-blocking mutations

• Dynamic PTM Regulation Studies:

  • Time-course experiments following stimuli

  • Inhibitor studies targeting specific modifying enzymes

  • Consider proximity labeling approaches to identify regulatory enzymes

• Quantitative PTM Analysis:

  • Use SILAC or TMT labeling for quantitative mass spectrometry

  • Develop standard curves with synthetic phosphopeptides/acetylpeptides

  • Compare stoichiometry across conditions

Methodological note: When analyzing PTMs, sample handling is critical - include phosphatase inhibitors, deacetylase inhibitors, and deSUMOylase inhibitors in lysis buffers to preserve modification status .

How can researchers leverage MTA2 antibodies for studying chromatin dynamics and 3D genome organization?

Investigating MTA2's role in chromatin dynamics and 3D genome architecture requires integrating antibody-based approaches with advanced genomic technologies:

• Chromatin Immunoprecipitation Methodologies:

  • Standard ChIP with MTA2 antibodies followed by qPCR or sequencing

  • ChIP-seq to map genome-wide binding profiles

  • CUT&RUN or CUT&Tag for improved signal-to-noise ratio

  • Validated MTA2 antibodies are suitable for these applications

• 3D Genome Architecture Analysis:

  • ChIA-PET using MTA2 antibodies to identify long-range chromatin interactions

  • HiChIP to combine Hi-C with MTA2 ChIP for interaction mapping

  • Integrate with ATAC-seq to correlate MTA2 binding with chromatin accessibility

  • 4C-seq focused on MTA2-bound regions as viewpoints

• Multi-Omics Integration:

  • Combine MTA2 ChIP-seq with:

    • RNA-seq to correlate binding with gene expression

    • Histone modification ChIP-seq to understand chromatin state

    • DNA methylation analysis for epigenetic context

  • Requires computational integration of multiple datasets

• Live-Cell Chromatin Dynamics:

  • Adaptation of MTA2 antibodies for live-cell imaging:

    • Nanobody development based on validated antibodies

    • Cell-permeable antibody fragments

  • Alternative: CRISPR-based tagging of endogenous MTA2

• Super-Resolution Microscopy Applications:

  • Immunofluorescence with MTA2 antibodies (66195-1-Ig at 1:1000-1:2000)

  • Apply STORM, PALM, or STED microscopy

  • Co-visualization with other chromatin components

  • Quantitative analysis of clustering and distribution

Technical consideration: For ChIP applications with MTA2 antibodies, crosslinking conditions are critical - standard 1% formaldehyde may be insufficient for proteins in large complexes like NURD/NuRD; consider testing dual crosslinkers (formaldehyde plus disuccinimidyl glutarate) for improved efficiency .